Diarrheal E. coli strains are listed the priority category B pathogen by the National Institute of Health (NIH). Enterotoxigenic Escherichia coli (ETEC) are the most common cause of traveler's diarrhea. The World Health Organization (WHO) reported that each year ETEC strains cause 380-500 million diarrhea cases to children and 400 million more cases to adult travelers, which results in approximately 300,000- 500,000 deaths annually. It is a top priority for WHO and other global and regional health programs to develop a broadly protective vaccine against ETEC-associated traveler's diarrhea. Currently, there are no effective vaccines available to protect against traveler's diarrhea. The virulence determinants of ETEC in diarrhea are bacterial adhesins and enterotoxins. Adhesins mediate bacteria attachment and colonization at small intestines. Enterotoxins disrupt fluid homeostasis in host epithelial cells that leads to fluid hyper-secretion and diarrhea. A vaccine inducing anti-adhesin immunity to block ETEC attachment and colonization and also antitoxin immunity to neutralize enterotoxicity can effectively protect against ETEC traveler's diarrhea. Our long-term goal is to understand molecular pathogenesis of diarrhea diseases and to develop disease prevention strategies. Our immediate objective is to develop a broadly protective vaccine against ETEC diarrhea. We here present preliminary data showing that: 1) a multiepitope ETEC CFA antigen carrying representative epitopes of 7 adhesins induced broadly protective immune responses; 2) LT and STa toxoid fusion antigens elicited neutralizing antibodies against both toxins; and 3) a porcine-type CFA-toxoid fusion induced protective immunity against heterogeneous adhesins and also the toxin. The specific hypotheses for this proposed study are that: 1) a multiepitope CFA- toxoid antigen will induce broad immune responses to 7 adhesins expressed by the most prevalent and most virulent ETEC strains and also to both LT and STa toxins; and 2) immunity induced by this multiepitope CFA-toxoid antigen will protect against adherence of ETEC strains expressing these 7 adhesins and also neutralize against both ETEC toxins, and this multiepitope CFA-toxoid fusion antigen can be developed as a subunit vaccine against ETEC traveler's diarrhea. Using innovative strategies to construct a CFA-toxoid fusion, mouse immunization studies and a novel piglet challenge study, the following specific aims are proposed to test our hypothesis: 1) To construct a multiepitope CFA-toxoid antigen for immunity against 7 ETEC adhesins and both toxins; 2) To evaluate this multiepitope CFA-toxin antigen for subunit vaccine candidacy against ETEC traveler's diarrhea. Results from this study will change current ETEC vaccine development strategies, of which multiple killed or live E. coli strains and recombinant toxin subunit protein are mixed together to induce anti-adhesin immunity. Mixing multiple strains in a product has been shown to result in lower immune responses (with no significant protection) and to cause adverse effects due to excessive somatic antigens and LPS included in products. Expressing antigenic epitopes of the most prevalent and virulent ETEC adhesins and both toxins as a single antigen can lead to a safe and effective subunit vaccine for broad protection against traveler's diarrhea.